2. Epigenetics
  3. Protein Arginine Deiminase
  4. PAD4-IN-2 TFA

PAD4-IN-2 TFA is a highly tumor-targeted and irreversible PAD4 inhibitor with an IC50 of 1.94 μM. PAD4-IN-2 TFA selectively recognizes sialic acid on tumor surfaces and accumulates in tumor tissues, with distribution in the cytoplasm of tumor cells and nuclei of neutrophils. PAD4-IN-2 TFA inhibits the PAD4-H3cit-NETs (Neutrophil Extracellular Traps) pathway, reduces senescent tumor-associated neutrophils, and promotes M1 macrophage polarization. The compound exhibits potent anti-tumor and anti-metastatic activities and is suitable for breast cancer research.

For research use only. We do not sell to patients.

PAD4-IN-2 TFA

PAD4-IN-2 TFA Chemical Structure

Size Price Stock Quantity
Solid + Solvent (Highly Recommended)
10 mM * 1 mL in DMSO
ready for reconstitution
 In-stock
Solution
10 mM * 1 mL in DMSO In-stock
Solid
5 mg In-stock
10 mg In-stock
25 mg In-stock
50 mg   Get quote  
100 mg   Get quote  

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Other Forms of PAD4-IN-2 TFA:

PAD4-IN-2 TFA Related Antibodies

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  • Biological Activity

  • Purity & Documentation

  • References

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Description

PAD4-IN-2 TFA is a highly tumor-targeted and irreversible PAD4 inhibitor with an IC50 of 1.94 μM. PAD4-IN-2 TFA selectively recognizes sialic acid on tumor surfaces and accumulates in tumor tissues, with distribution in the cytoplasm of tumor cells and nuclei of neutrophils. PAD4-IN-2 TFA inhibits the PAD4-H3cit-NETs (Neutrophil Extracellular Traps) pathway, reduces senescent tumor-associated neutrophils, and promotes M1 macrophage polarization. The compound exhibits potent anti-tumor and anti-metastatic activities and is suitable for breast cancer research[1].

IC50 & Target

IC50: 1.94 μM (PAD4)[1].
In Vitro

PAD4-IN-2 (compound 5i) TFA inhibits PAD4 enzyme activity in a colorimetric assay (IC50 = 1.94 μM) and exhibits negligible cytotoxicity in LLC, 4T1, and normal HL7702 cells after 48 h treatment (IC50 > 100 μM)[1].
PAD4-IN-2 (25-100 μM; 48 h) TFA effectively inhibits the monoclonal proliferation and dose-dependently suppresses the migration of 4T1 cells[1].
PAD4-IN-2 (50–100 μM; 1–48 h) TFA is selectively taken up by 4T1 cells in a time-dependent manner but not by normal HL7702 cells, induces mitochondrial shrinkage and pseudopodia reduction, localizes to neutrophil nuclei to decrease histone H3 citrullination (H3cit) levels, and inhibits neutrophil extracellular trap (NET) formation[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

PAD4-IN-2 TFA Related Antibodies

Cell Viability Assay[1]

Cell Line: LLC, 4T1, and HL7702 cells
Concentration: 3.125, 6.25, 12.5, 25, 50, and 100 μM
Incubation Time: 48 h
Result: Showed no significant lethal effect on either tumor or normal cells, with an IC50 value greater than 100 μM.

Cell Proliferation Assay[1]

Cell Line: 4T1 cells
Concentration: 25, 50, and 100 μM
Incubation Time: 48 h
Result: Reduced the number of single cell proliferation significantly and effectively inhibited the monoclonal proliferation of tumor cells.

Cell Migration Assay [1]

Cell Line: 4T1 cells
Concentration: 25, 50, and 100 μM
Incubation Time: 48 h
Result: Inhibited tumor cell migration in a dose-dependent manner, and significantly decreased both the number of migrated cells and the migration distance.

Immunofluorescence[1]

Cell Line: 4T1 cells and bone marrow neutrophils
Concentration: 50 μM and 100 μM
Incubation Time: 48 h
Result: Localized to the cytoplasm of tumor cells and the nucleus of neutrophils, decreased intracellular histone 3 citrullination (H3cit) levels, and significantly inhibited the formation of neutrophil extracellular traps (NETs).
In Vivo

PAD4-IN-2 (compound 5i) TFA (6.18 mg/kg; i.v. ; every day for 21 days) inhibits primary tumor growth and suppresses lung metastasis in an orthotopic 4T1 breast cancer mouse model[1].
PAD4-IN-2 (6.18 mg/kg; i.p. ; once daily for 7 days) TFA shows initial antitumor efficacy in an S180 sarcoma mouse model[1].
PAD4-IN-2 (1.24-6.18 mg/kg; i.v. ; every day for 21 days) TFA decreases NETs formation, alters immune cells infiltration in the tumor microenvironment, and exhibits no significant hepatotoxicity or nephrotoxicity in vivo[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Model: S180 sarcoma mouse model (Kunming mice)[1]
Dosage: 6.18 mg/kg
Administration: Intraperitoneal injection (i.p.), once daily for 7 days
Result: Inhibited tumor growth with an in vivo tumor inhibition rate (TIR) of 49.2%.
Animal Model: Orthotopic 4T1 breast cancer mouse model (BALB/c female mice)[1]
Dosage: 1.24, 3.09, and 6.18 mg/kg
Administration: Tail vein intravenous injection (i.v.), once daily for 21 days
Result: Reduced primary tumor volume and tumor weight substantially at the dose of 10 μmol/kg.
Suppressed the number of metastatic tumor nodules in the lungs markedly in a dose-dependent manner.
Maintained stable body weight in treated mice throughout the administration period.
Animal Model: Orthotopic 4T1 breast cancer mouse model (BALB/c female mice)[1]
Dosage: 6.18 mg/kg
Administration: Tail vein intravenous injection (i.v.), once daily for 21 days
Result: Showed intact alveolar structures and absence of tumor cell lesions in H&E-stained lung sections.
Decreased the number of Ly6G-positive neutrophils and H3cit-positive cells in immunofluorescence-stained tumor sections.
Caused no significant variations in serum levels of Cr, BUN, AST, and ALT compared with the normal saline control group.
Exhibited no apparent histopathological damage or organic lesions in H&E-stained sections of the heart, liver, spleen, and kidney tissues.
Reduced the proportion of aged neutrophils (Naged CD194hi CD62Llo) in tumor tissues.Increased the ratio of M1-type macrophages in tumor tissues.
Molecular Weight

617.73

Formula

C22H24BClF3N7O8
Appearance

Solid

Color

Yellow to orange

SMILES

O=C(NCC1=CC=CC(B(O)O)=C1)[C@H](CCCNC(CCl)=N)NC2=CC=C([N+]([O-])=O)C3=NON=C32.O=C(C(F)(F)F)O
Shipping

Room temperature in continental US; may vary elsewhere.
Storage

4°C, sealed storage, away from moisture

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

Solvent & Solubility
In Vitro: 

DMSO : 100 mg/mL (161.88 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 1.6188 mL 8.0942 mL 16.1883 mL
5 mM 0.3238 mL 1.6188 mL 3.2377 mL
View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

  • Molarity Calculator

  • Dilution Calculator

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

Mass
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Concentration
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Volume
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Molecular Weight *

Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2

Concentration (start)

C1

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Volume (start)

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Concentration (final)

C2

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Volume (final)

V2

In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

    Solubility: ≥ 2.5 mg/mL (4.05 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

    Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
  • Protocol 2

    Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

    Solubility: ≥ 2.5 mg/mL (4.05 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

    Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:

Dosage

mg/kg

Animal weight
(per animal)

g

Dosing volume
(per animal)

μL

Number of animals

Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
%
DMSO +
% 
+
%
Tween-80 +
%
Saline
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Calculation results:
Working solution concentration: mg/mL
Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
 If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Purity & Documentation
References

Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
DMSO 1 mM 1.6188 mL 8.0942 mL 16.1883 mL 40.4708 mL
5 mM 0.3238 mL 1.6188 mL 3.2377 mL 8.0942 mL
10 mM 0.1619 mL 0.8094 mL 1.6188 mL 4.0471 mL
15 mM 0.1079 mL 0.5396 mL 1.0792 mL 2.6981 mL
20 mM 0.0809 mL 0.4047 mL 0.8094 mL 2.0235 mL
25 mM 0.0648 mL 0.3238 mL 0.6475 mL 1.6188 mL
30 mM 0.0540 mL 0.2698 mL 0.5396 mL 1.3490 mL
40 mM 0.0405 mL 0.2024 mL 0.4047 mL 1.0118 mL
50 mM 0.0324 mL 0.1619 mL 0.3238 mL 0.8094 mL
60 mM 0.0270 mL 0.1349 mL 0.2698 mL 0.6745 mL
80 mM 0.0202 mL 0.1012 mL 0.2024 mL 0.5059 mL
100 mM 0.0162 mL 0.0809 mL 0.1619 mL 0.4047 mL
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PAD4-IN-2 TFA Related Classifications

Help & FAQs
  • Do most proteins show cross-species activity?

    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

Keywords:

PAD4-IN-2Protein Arginine DeiminasePeptidylarginine DeiminasePAD4 inhibitorPhenylboronic acidSialic acidNETsH3citTumor microenvironmentMetastasisLLC4T1HL7702NeutrophilsMacrophagesS180 sarcoma modelOrthotopic 4T1 breast cancer modelBreast cancerSarcomaLung metastasisInhibitorinhibitorinhibit

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